Treatment of cancer with human chorionic gonadotropin

ABSTRACT

Methods useful for treating cancers are disclosed. The methods involve administering human chorionic gonadotropin (hCG) or human luteinizing hormone (hLH) to patients having cancers. Articles of manufacture that are useful for carrying out the described methods are also described. The claimed methods are effective against breast, prostate, ovary, and stomach carcinomas, as well as neuroblastomas, and Kaposi&#39;s sarcoma, among others.

BACKGROUND OF THE INVENTION

Treatment of tumors, including cancers, is an area of clinical medicinethat remains fraught with complications and often presents an array ofsuboptimal treatment choices. A major problem in treating cancer is thatmost or all of the known therapies have serious adverse side effects.For example, surgery is commonly employed to remove a cancer from apatient. However, surgery often disfigures the patient or interfereswith normal bodily functions. Other treatments such as chemotherapy andradiation treatment also cause undesired side effects.

Kaposi's sarcoma (KS) is one example of a cancer that is difficult totreat. Kaposi's sarcoma is the most common malignancy in patients withacquired immunodeficiency syndrome (AIDS) in the United States. KSoccurs in approximately 30% of male AIDS patients, but only 3% of femaleAIDS patients Elford et al. (1991) AIDS 7: 1667-1671!. The exactmechanism by which KS develops is unknown. Kaposi's sarcoma is amultifocal neoplasm, consisting of several cell types and abundantangiogenesis. The tumor cell is believed to be a spindle-shaped cell andis usually considered to be of endothelial origin Rutgers et al. (1986)Am. J. Pathol. 122: 493-499!. However, its precise cellular origin andeven its classification as a true monoclonal malignancy versus apolyclonal hyperplasia have remained elusive Shaw et al. (1984) Science226: 1165-1171; Holden et al. (1989) J. Invest. Dermatol. 93: 119S-124S;Hashimoto et al. (1987) Pathol. Res. Pract. 182: 658-668!.

Existing Kaposi's sarcoma treatments often cause myelotoxicity andneurotoxicity see, e.g. Northfeldt et al. (1991) Hematology/OncologyClinics of North America 5: 297-310, which is incorporated herein byreference!. Presently available Kaposi's sarcoma therapies can alsoinduce immunosuppression, compounding the pre-existing immunodeficiencythat is usually present in AIDS patients.

Thus, a great need exists for a treatment for Kaposi's sarcoma and othercancers that does not cause severe adverse side effects. Hormonaltreatments offer promise of such a treatment.

Many cancers secrete hormones such as human chorionic gonadotropin (hCG)and/or an hCG subunit. Indeed, elevated hCG serum concentration isconsidered a reliable indicator of the presence of some tumors see,e.g., Bagshawe (1992) Acta Oncol. 31: 99-106; Stenman et al. (1993)Scand. J. Clin. Lab. Invest. Suppl. 216: 42-78; Mann et al. (1993)Scand. J. Clin. Lab. Invest. Suppl. 216: 97-104!. Elevated serum levelsof hCG or an hCG subunit are found in patients having gestationaltrophoblastic tumors and testicular germ cell tumors, as well asnongonadal and nontrophoblastic tumors such as cancers of the bladder,pancreas, cervix, lung, liver, and stomach Bidart et al. (1993) TEM 4:285-291; Marcillac et al. (1992) Cancer Res. 52: 3901-3907; Alfthan etal. (1992) Cancer Res. 52: 4628-4633!.

Human chorionic gonadotropin belongs to a family of glycoproteinhormones, human luteinizing hormone (lutropin, hLH), follitropin (FSH),and thyrotropin (TSH). Each of these hormones is composed of twodissimilar, noncovalently bound subunits, α and β. The hormones share acommon α subunit, while the β subunits differ slightly in length andamino acid sequence Ryan et al. (1988) FASEB J. 2: 2661-2669; Ward etal. in Reproduction in Domestic Animals, 4th ed., Cuppos, PT, ed., pp.25-80, Academic Press, NY (1991)!. The most closely related of the βsubunits are those of hCG and hLH, which are 85% identical, except foran approximately 20 amino acid extension on the carboxy terminus of hCG.Indeed, hCG and hLH act through a common receptor Loosfelt et al. (1989)Science 245: 525-528; McFarland et al. (1989) Science 245: 494-499! andelicit identical biological responses Pierce and Parsons (1981) Ann.Rev. Biochem. 50: 466; Strickland et al. in Luteinizing hormone actionand receptors, M. Ascoli, Ed., CRC Press, Boca Raton Fla., 1985, p. 1!

The present invention is based on the discovery that hCG and relatedhormones are useful not only as a marker for detecting cancers, but alsoas a treatment for cancers. Provided herein are an effective methods fortreating cancers using human hormones such as human chorionicgonadotropin or human lutropin, and articles of manufacture that areuseful for carrying out these methods.

SUMMARY OF THE INVENTION

The invention provides methods for treating a cancer in a patient. Themethods involve administering to the patient a therapeutically effectiveamount of a pharmaceutical agent selected from the group consisting ofhuman chorionic gonadotropin, human lutropin (hLH), a β subunit of hCGor hLH, and a biologically active fragment of hCG, hLH, or a β-subunitof hCG or hLH. Also provided are articles of manufacture that includepackaging material and a pharmaceutical agent contained within thepackaging material, wherein the pharmaceutical agent is therapeuticallyeffective for treating a cancer and is selected from the groupconsisting of human chorionic gonadotropin (hCG), human lutropin (hLH),a β-subunit of hCG or hLH, and a biologically active fragment of hCG,hLH, or a β-subunit of hCG or hLH. The packaging material includes alabel which indicates that the pharmaceutical agent is useful fortreating a malignant neoplastic disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 show that tumor cells undergo apoptosis when treated with hCG.The experimental protocol is as described in Example 6. A) FIG. 1A Tumorfrom a mouse that had not been treated with hCG has many mitotic figureswhen stained with the hematoxylin and eosin stains. See, e.g.,Biological Staining Methods, George D. Jurr Div., Baird & Tatlock,Romford UK (1969)!. B) FIG. 1B Thin tissue section of a tumor from amouse that had been treated with hCG (100 USP units injectedsubcutaneously daily for 7 days) shows areas that have few cells, andother areas exhibit dense nuclear masses. 1C, 1D Tumors from hCG-treatedmice were stained in situ to detect the presence of DNA fragmentation.Apoptotic cells are brown, while normal viable cells appear blue afterstaining.

FIG. 2 shows the results of chemical crosslinking experiments in whichradiolabelled β-hCG was cross-linked to the hCG receptor, after whichcells were lysed and proteins extracted. Lane 1: Kaposi's sarcoma cellstrain KS-4 (passage 9); Lane 2: Kaposi's sarcoma cell line KS Y-1(passage 35); Lanes 3-7: Kaposi's sarcoma cell lines from HIV positivepatients; Lane 8: primary colon carcinoma cells from patient #1539.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Definitions

A cell line is said to be "malignant" if, when the cell line is injectedinto a host animal, the host animal develops tumors or cancers that areanaplastic, invasive, and/or metastatic. A "human" tumor is comprised ofcells that have human chromosomes. Such tumors include those in a humanpatient, and tumors resulting from the introduction of a human malignantcell line into a non-human host animal if cells from such tumors havehuman chromosomes. A tumor is said to be "long-lasting" when the tumorpersists in an animal for at least about one month.

The term "effective amount" means a dosage sufficient to produce adesired result. The desired result can be subjective or objectiveimprovement in the recipient of the dosage, a decrease in tumor size, adecrease in the rate of growth of cancer cells, or a decrease inmetastasis.

The terms "treating cancer", "therapy", and the like mean generally atreatment that causes any improvement in a mammal having a cancerwherein the improvement can be ascribed to treatment with the hormonepolypeptide. The improvement can be either subjective or objective. Forexample, if the mammal is human, the patient may note improved vigor orvitality or decreased pain as subjective symptoms of improvement orresponse to therapy. Alternatively, the clinician may notice a decreasein tumor size or tumor burden based on physical exam, laboratoryparameters, tumor markers, or radiographic findings.

Some laboratory signs that the clinician may observe for response totherapy include normalization of tests such as white blood cell count,red blood cell count, platelet count, erythrocyte sedimentation rate,and various enzyme levels such as transaminases and hydrogenases.Additionally, the clinician may observe a decrease in a detectable tumormarker such as prostatic specific antigen (PSA) or chorio embryonicantigen (CEA). Alternatively, other tests can be used to evaluateobjective improvement such as sonongrams, nuclear magnetic resonancetesting and positron emissions testing.

"Inhibiting the growth of cancer cells" can be evaluated by any acceptedmethod of measuring whether growth of the cancer cells has been slowedor diminished. This includes direct observation and indirect evaluationsuch as subjective symptoms or objective signs as discussed above.

A "hormone-responsive cancer" refers to a cancer that responds totreatment with hCG, hLH, a β-subunit of hCG or hLH, or a biologicallyactive subfragment. To determine whether a cancer is hormone-responsive,the clinician can test whether hormone treatment inhibits growth ofcells derived from a cancer of that particular type. Appropriate assaysare described below.

A "biologically active fragment" of hCG, hLH, or a β-subunit of hCG orhLH exhibits a biological activity of the indicated hormone. Todetermine whether a polypeptide fragment is biologically active, one canperform an assay to detect a biological effect that is typical of cellsexposed to hCG or hLH, such as stimulation of progesterone or cAMPproduction. Alternatively, one can test the fragment to determinewhether it binds to the lutropin-choriogonadotropin receptor (LH-CG-R).Suitable tests are described below.

Hormone Polypeptides Useful for Treating Cancer

Hormone polypeptides that are useful in the claimed articles ofmanufacture and methods for treating cancer include both hCG and hLH.Furthermore, the β-subunits of hCG and hLH are useful whether or notcomplexed to the α-subunit that hCG and hLH share. Also, biologicallyactive fragments of β-hCG and β-hLH also function to treat canceraccording to the present invention.

hCG and hLH are available from several sources. For example, hCG and hLHare available commercially from Sigma Chemical Co. (St. Louis, Mo.). hCGis available in a form suitable for therapeutic use from Wyeth-AyerstLaboratories (APL™, Philadelphia Pa.), Organon, Inc. (Pregnyl™, WestOrange, N.J.), and Serono Laboratories, Inc. (Profasi™, Randolph Mass.).

Alternatively, one can isolate the natural hCG and hLH polypeptides fromhuman cells by conventional techniques, such as affinity chromatography.Conveniently, polyclonal or monoclonal immunoglobulins obtained againsthCG or hLH can be used to prepare a suitable affinity column by wellknown techniques see, e.g., Hudson and May, Practical Immunology,Blackwell Scientific Publications, Oxford, United Kingdom, 1980, whichis incorporated herein by reference!.

One can also produce the hormone polypeptides that are useful in thepresent invention by chemical or enzymatic synthesis. Techniques forsolid phase chemical synthesis of polypeptides are described, forexample, in Merrifield, J. Amer. Chem. Soc. 85: 2149-2156 (1963), whichis incorporated herein by reference. Such chemical synthesis isgenerally employed for the production of polypeptides of fewer thanabout 100 amino acids, more usually fewer than about 80 amino acids, andtypically fewer than about 50 amino acids.

A preferred method for producing the hormone polypeptides of theinvention involves recombinant expression. For this purpose, natural orsynthetic nucleic acids that code for the hCG or hLH, or the β-subunitof either hormone, or an active fragment of either hormone, willtypically be operably linked to a promoter to form an expressioncassette. The α- and β-hCG cDNAs have been cloned and the nucleotidesequences determined Xia, H., J. Molecular Endocrinology, pp. 337-343(Jun. 10, 1993); Sherman, G. B., J. Molecular Endocrinology, pp. 951-959(Jun. 6, 1992); Segal, S. J. (ed.), Plenum Press, N.Y., pp. 37-51(1980); Basic and Clinical Endocrinology, Gieseman, L. K., (ed.), Ch.20, pp. 543-567 (1991); Ward et al., supra.!. Therefore, one of skillcan readily identify and clone the cDNAs that code for thesepolypeptides. Alternatively, one can synthesize the desired codingregions chemically.

To produce the hormone polypeptides, one introduces an expressioncassette that codes for the hormone polypeptide into an appropriate hostcell. Suitable host cells include yeast, filamentous fungi, insects(especially employing baculoviral vectors), and mammalian cells, as wellas bacterial systems.

Mammalian or insect cell expression systems are preferred, since proteinfolding, transport and processing (including glycosylation) closelyapproximate that which occurs in the human see, e.g., E. Winnacker, FromGenes to Clones, VCH Publishers, New York (1987), which is incorporatedherein by reference!. For examples of suitable expression systems forthe hormone polypeptides useful in the present invention, see, e.g.,Corless et al. (1987) J. Biol. Chem. 262: 14187-14203; Lustbader et al.(1987) J. Biol. Chem. 262: 14204-14212; Huang et al. (1993) Mol. Cell.Endocrinol. 90: 211-218; Reddy et al, (1985) Proc. Natl. Acad. Sci. USA8: 3644-3648; all of which are incorporated herein by reference.

The baculovirus expression system is most preferred, producing thehormone polypeptides in relatively high yields, and in an active form.See, e.g., Nakhai et al. (1992) Indian J. Biochem & Biophys. 29:315-321.

Once expressed, one can purify the hormone polypeptides from lysed cellsor, preferably, from culture medium into which the hormone polypeptidesare secreted. Standard procedures of the art are suitable, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), which is incorporated hereinby reference). Substantially pure hormones of at least about 90 to 95%homogeneity are preferred, and those of 98 to 99% or greater homogeneitymost preferred, for pharmaceutical uses.

Once purified, partially or to homogeneity as desired, the hormones canthen be used therapeutically.

It is recognized that amino acid residues in the hormone polypeptidesmay be replaced by other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity). Because the substitutedamino acids have similar properties, the substitutions do not change thefunctional properties of the polypeptides. The following six groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Identifying Biologically Active Hormone Fragments

Hormone polypeptides that are effective against cancers include theintact hCG and hLH dimers, the β-subunits of hCG and hLH, andsubfragments of the hCG and hLH β-subunits. The hCG and hLH α-subunitsalone are not effective. Among the biologically active subfragments ofβ-hCG that are effective against cancer are β-hCG(109-145) Stevens(1986) CIBA Foundation Symp. 19: 200-225! and β-hCG(109-119) Lyer et al.(1992) Int. J. Peptide Prot. Res. 39: 137-192!. These latterpolypeptides are available commercially from Bachem Bioscience, Inc.(King of Prussia, Pa.), and β-hCG(109-145) is also available from SigmaChemical Co.

So long as the polypeptide or subfragment retains biological activity,it can be used in the claimed cancer treatment methods. To determinewhether a particular polypeptide is biologically active, cells thatexpress the hLH-hCG receptor (LH-CG-R) are exposed to the polypeptidefragment being tested, after which the cells are assayed for biologicaleffects that are indicative of hCG or hLH presence. One can conduct thisassay using a mammalian cell line that naturally expresses the LH-CG-R.One such cell type that is useful for this assay is the MA-10transformed murine Leydig cell line Ascoli (1981) Endocrinology 108:88-95!. The MA-10 cells are grown as described by Chen and Puett (1991a)J. Biol. Chem. 266: 6904-6908; Chen and Puett (1991b) Biochemistry 30:10171-10175; Chen et al. (1991) J. Biol. Chem. 266: 19357-19361!.

As an alternative to cells that naturally express LH-CG-R, one can usecells that express LH-CG-R because the cells have been transfected withan expression vector that harbors the LH-CG-R gene or cDNA. A suitableexpression vector and cell line, as well as the nucleotide sequence ofthe LH-CG-R cDNA, are described in McFarland et al. (1989) Science 245:494-499.

LH-CG-R-expressing cells exposed to a biologically active fragment ofhCG or hLH will have elevated concentrations of cyclic AMP (cAMP). cAMPassays are described in, for example, McFarland et al., supra., Ascoliet al. (1989) J. Biol. Chem. 264: 6674, and Segaloff and Ascoli (1981)J. Biol. Chem. 256: 11420. A biologically active hormone fragment willcause cells treated with 10 ng/ml of the fragment for 15 minutes at 37°C. to have cAMP levels at least about 1.5 times as great as cells nottreated with the fragment.

Another commonly used assay is to determine whether cells treated withthe polypeptide produce higher levels of progesterone than untreatedcells. The hormone polypeptide is added at various concentrations to thecells in a suitable medium. After a four hour incubation at 37°,progesterone is measured by radioimmunoassay. Basal progesteroneconcentration for untreated MA-10 cells is typically less than 10 ng/ml,while cells incubated in the presence of a biologically active hormonepolypeptide will typically produce at least 500 ng/ml progesterone Huanget al. (1993) J. Biol. Chem. 268: 9311-9315!.

An alternative assay to assess whether a hCG or hLH polypeptide orsubfragment is biologically active is to determine whether thepolypeptide binds to the cellular receptor for hCG and hLH. A suitableassay is described in Huang et al., supra.

Testing for Efficacy Against a Particular Cancer

The claimed methods and articles of manufacture are effective against arange of different cancer types. For example, many neoplasms areamenable to treatment using the claimed invention. These neoplasmsinclude gestational trophoblastic tumors and testicular germ celltumors, as well as nongonadal and nontrophoblastic tumors such ascancers of the breast, bladder, pancreas, cervix, lung, liver, ovary,colon, and stomach. Patients having these types of cancers typicallyexhibit elevated serum levels of hCG and/or/ β-hCG. See, e.g., Bidart,supra; Marcillac et al., supra.; and Alfthan et al., supra.

The invention is also useful for treating neuroblastomas and Kaposi'ssarcoma. Cancers that are particularly susceptible to the claimedtreatment methods are Kaposi's sarcoma, neuroblastoma, and carcinomas ofthe breast, prostate, ovary, and stomach.

The clinician can test the efficacy of hCG against a particular tumortype, either in vitro or in vivo. For in vitro tests, cells derived fromthe tumor are grown in the presence or absence of the hormone and theeffect of the hormone is determined. One commonly utilized assay fortumor cell growth is the methylcellulose assay Lunardi-Iskandar et al.(1985) Clin. Exp. Immunol. 60: 285-293!. The cells are plated in mediumcontaining methylcellulose, which prevents nontumor cells fromundergoing mitosis and forming colonies. The hormone polypeptides of theinvention will prevent tumor cells from forming as many colonies asuntreated cells. Another means for measuring the inhibitory effect ofthe hCG of hLH polypeptides is by measuring the rate of incorporation ofradiolabelled metabolites such as tritiated thymidine.

The growth of a cell line is said to be "inhibited" by a hormonetreatment if, when assayed by means such as radioisotope incorporationinto the cells, the treated cells proliferate at a rate that is lessthan about 80% of the proliferation rate of untreated control cells, andpreferably less than about 70% of the untreated cell proliferation rate.More preferably, the growth rate is inhibited by at least 50%. If growthis assayed by a means such as plating in methylcellulose, the growth ofa cell line is said to be "inhibited" if the treated cells give rise toless than about 80% of the number of colonies that grow from a likenumber of untreated cells. Preferably, the number of colonies fromtreated cells is less than about 70% of the number from untreated cells.More preferably, the number of colonies is decreased by at least 50%.

In addition to, or instead of, testing a hormone for efficacy against aparticular tumor cell type in vitro, the clinician can test the hormonein vivo. For in vivo tests, cells derived from the tumor type areinjected into laboratory animals such as immunodeficient mice.Typically, either the laboratory animals or the cells have beenpre-treated with the hormone. The animals are then monitored todetermine whether tumors arise at the site of injection, or elsewhere inthe animal.

One method for carrying out these in vivo tests is as follows. The tumorcells to be tested are grown for 48 hours in RPMI1640+10% fetal calfserum in the presence of 100 USP units/ml hCG per ml. As a control,tumor cells are grown in the absence of hCG. Approximately 5×10⁶ cellsare then subcutaneously injected into SCID or nude mice (Beige, BALB/c,Swiss, or NCr see, e.g., Croyba et al. (1993) Laboratory Animal Science,43: 120-122)!. Alternatively, the efficacy of a hormone against a tumorcell type can be tested by inoculating a mouse with hCG before challengewith the tumor cells, rather than pretreating the cells. For example,mice can be injected with 10-100 USP units hCG daily for 5-7 days beforechallenge with the tumor cells. One can test mice of either sex, and atvarious ages (neonate, young, adult).

Laboratory animals that are either treated with a hormone polypeptideprior to tumor cell inoculation, or inoculated with tumor cells thathave been grown in the presence of the hormone polypeptide, typicallywill not develop tumors. For example, in the absence of hCGpretreatment, Kaposi's sarcoma cells will typically induce a strongangiogenic reaction at the site of inoculation within seven days. Tumorswill usually develop and persist for at least three months. These tumorscan occur at the site of injection and also as metastases in one or moreof the lung, spleen, skin, or pancreas. Typically, tumors induced by theKS cell lines will metastasize.

Mice that are injected with Kaposi's sarcoma cells that have beenpretreated with hCG, or mice that have been pre-inoculated with hCGbefore challenge with the Kaposi's sarcoma cells, do not develop tumorswithin two months post-challenge.

Cancer Therapy Methods and Articles of Manufacture

Pharmaceutical compositions containing the hormone polypeptidesdescribed herein are administered to an individual having cancer, Intherapeutic applications, compositions are administered to a humanpatient in an amount sufficient to cause regression of the tumor, or atleast partially arrest the tumorigenesis and metastasis. An amountadequate to accomplish this is defined as a "therapeutically effectivedose." Amounts effective for this use will depend on, e.g., the natureof the hormone (specific activity, etc.), the manner of administration,the stage and severity of the cancer, the weight and general state ofhealth of the patient, and the judgment of the prescribing physician.Typically, doses will range from about 3000 to about 5000 USP units perpatient, (70 kg) per day of hormone polypeptide per day. If a cancer hasmetastasized a larger dose can be employed. Generally, the dose will berepeated daily until the tumors are gone. Typically, a minimum of one totwo weeks of treatment is required.

Single or multiple administrations of the hormone polypeptidecompositions can be carried out with dose levels and pattern beingselected by the treating physician. In any event, the pharmaceuticalformulations should provide a quantity of hormone sufficient toeffectively treat the patient. Administration should begin at the firstindication of undesirable cellular proliferation or shortly afterdiagnosis, and continue until symptoms are substantially abated and fora period thereafter. In well established cases of cancer, loading dosesfollowed by maintenance doses will be required.

The clinician can monitor whether a dosage regime is correct bymonitoring the concentration of β-hCG in the patient's blood plasma. Theexperimental data presented herein allows one to ascertain the properlevel of β-hCG. As shown in Example 2, serum from human females who areat an early stage in pregnancy inhibits tumor cell growth moreeffectively than serum from late-pregnancy females. During the earlyweeks of pregnancy, β-hCG levels increase rapidly, reaching a peak of160 USP units per ml plasma during the tenth gestational week. β-hCGlevels then decline gradually to about 10 USP units per ml in the thirdtrimester of pregnancy. Therefore, regardless of the route ofadministration, the clinician should adjust the dosage regime asrequired to maintain plasma β-hCG concentration at a level of at least10 USP units per ml, and preferably within a range of 100 USP units perml to 200 USP units per ml. One can assess these levels using the assaysdescribed above, or by other methods such as a competitiveradioimmunoassay.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Themost preferred route of administration is intralesional or intramuscularinjection. The invention provides compositions for parenteraladministration which comprise a solution of the hormone dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions can be sterilized by conventional, well known sterilizationtechniques, or can be sterile filtered. The resulting aqueous solutionscan be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

A preferred composition of hCG is available commercially from severalmanufacturers (Wyeth-Ayerst Laboratories (APL™, Philadelphia Pa.),Organon, Inc. (Pregnyl™, West Orange, N.J.), and Serono Laboratories,Inc. (Profasi™, Randolph Mass.). These compositions are of sufficientpurity for therapeutic use. To prepare the hormone for therapeutic use,the clinician follows the directions provided by the manufacturer.Typically, this involves reconstituting the dried hormone polypeptide insterile water to obtain the desired concentration.

A typical pharmaceutical composition for intravenous infusion could bemade up to contain 250 ml of sterile Ringer's solution, and 3000-5000USP units of hormone polypeptide. Actual methods for preparingparenterally administrable compounds will be known or apparent to thoseskilled in the art and are described in more detail in for example,Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), which is incorporated herein by reference.

For solid compositions of the hormones of the invention, conventionalnontoxic solid carriers can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. For oral administration, a pharmaceuticallyacceptable nontoxic composition is formed by incorporating any of thenormally employed excipients, such as those carriers previously listed,and generally 10-95% of active ingredient, that is, one or moreanti-cancer hormones, more preferably at a concentration of 25%-75%.

The present invention also includes an article of manufacture thatcomprises at least one pharmaceutical agent contained within packagingmaterial. The pharmaceutical agent is therapeutically effective fortreating a malignant neoplastic disease and is selected from the groupof hormone polypeptides disclosed herein. This group consists of humanchorionic gonadotropin (hCG), human lutropin (hLH), a β-subunit of hCGor hLH, or an active fragment of one of these hormone polypeptides.

The packaging material comprises a label which indicates that thepharmaceutical agent can be used for treating a malignant neoplasticdisease. The packaging material will usually also include instructionsfor administering the pharmaceutical agent, as well as a list of cancersagainst which the pharmaceutical agent is effective.

The following examples are provided by way of illustration and are notintended to limit the scope of the claims.

EXAMPLE 1

Pregnant Mice are Resistant to Tumorigenesis Induced by Kaposi's SarcomaCell Line

To demonstrate that pregnant mice are resistant to tumorigenesis, weinjected Kaposi's sarcoma cells into mice at various stages ofpregnancy. 5×10⁷ Kaposi's sarcoma cells (KS-Y-1) were injected intoBG-nude or Scid immunodeficient mice. nude mice (Beige, BALB/c, Swiss,or NCr (see, e.g., Croyba et al. (1993) Laboratory Animal Science, 43:120-122). Mice were either early in pregnancy (≦10 days gestation), latein pregnancy (≧16 days gestation), or not pregnant. One month afterinoculation, mice were sacrificed and examined for the presence oftumors.

Results are shown in Table 1. None of the mice injected with Kaposi'ssarcoma cells while early in pregnancy developed tumors. In contrast,both of the non-pregnant mice developed metastatic tumors. Mice that hadbeen injected late in pregnancy developed tumors, although these tumorswere much smaller than those in non-pregnant mice, and did notmetastasize.

                  TABLE 1                                                         ______________________________________                                        Inhibition of tumor growth induced by KS Y-1 cell in immunodeficient          mice during pregnancy.                                                                Inoculation Tumor size                                                                              Angio-                                          Animal (#)                                                                            (Gestation day #)                                                                         (mm)      genesis                                                                              Metastasis                               ______________________________________                                        early stage                                                                   Bg (4)  6-10        0         -      -                                        Scid (3)                                                                              5-10        0         -      -                                        late stage                                                                    Bg (3)  16-18       1 × 1 to                                                                          +      -                                                            2 × 2                                               Scid (3)                                                                              16-18       1 × 1 to                                                                          +      -                                                            3 × 2                                               Bg      Non Pregnant                                                                              25 × 20                                                                           +      +                                        Scid    Non Pregnant                                                                              27 × 22                                                                           +      +                                        ______________________________________                                         5 × 10.sup.6 KS Y1 cells were injected intraperitoneally into early     (3 to 10 days) and late stage (14 to 17 days) pregnant immunodeficient        mice (Beigexid-nude: Bgnude; Scid: severe combined immunodeficient mice).     The mice were sacrificed 1 month postinoculation. Metastasis was defined      by the presence of characteristic tumors in lung, pancreas and colon.    

EXAMPLE 2

Serum from Pregnant Females Inhibits Growth of Kaposi's Sarcoma Cells invitro

This example demonstrates that serum from pregnant mouse and humanfemales inhibits the growth of tumor cells. We assayed the ability ofKaposi's sarcoma (KS Y-1) cells to grow in the presence of serumobtained from pregnant females. We tested both human and murine serum,obtained either early or late in pregnancy.

5×10⁶ cells/ml KS Y-1 were seeded in methylcellulose in the presence orabsence of serum from pregnant females. We utilized the method describedin Lunardi-lskandar et al. (1985) Clin. Exp. Immunol. 60: 285-293.Results are shown in Table 2. The number of colonies formed is inverselyproportional to the inhibitory effect of the pregnancy serum. Theseresults demonstrate that serum obtained from human or murine femalesearly in pregnancy is highly effective in inhibiting growth of Kaposi'ssarcoma cells in vivo. Late pregnancy serum is slightly less effectivethan early pregnancy serum, but nevertheless exhibits an inhibitoryeffect compared to serum from males and non-pregnant females.

                  TABLE 2                                                         ______________________________________                                        Sera of pregnant mice and humans inhibits in vitro colony formation of        KS Y-1 cells.                                                                            Serum-                                                             Source of  free    Early pregnancy*                                                                           Late pregnancy*                               Cells serum    0%      1%   5%   10%  1%   5%   10%                           ______________________________________                                        KS Y-1         191                                                                  mouse A          40   4    0    146  108  70                                  B                36   2    0    138  102  69                                  C                41   7    0    140  105  67                                  D                31   10   0    ND   ND   ND                                  Human A          23   11   3    169  142  92                                  B                21   12   5    165  102  74                                  C                34   7    0    167  121  75                                  D                32   13   3    157  120  73                            ATL1  310                                                                           mouse A          293  276  298  283  301  322                                 Human A          280  269  301  260  273  265                           ______________________________________                                                        Sera from non-                                                                pregnant individuals                                                                1%     5%   10%                                         ______________________________________                                        KS Y-1                                                                              Male            193    178  179                                               Non-pregnant female                                                                           177    170  175                                         ______________________________________                                         KS Y1 cells were seeded in a methylcellulose (0.8%, v/v) clonogenic assay     in the presence of an increasing percentage (v/v) of serum from pregnant      women or mice. Colonies (>50 cells) were counted following a 10 day           incubation period. Number of colonies are expressed as mean number of         colonies per 5 × 10.sup.4 cells seeded in triplicate cultures. The      standard deviation did not exceed 10% of the mean values. ATL 1 is an         human adult Tcell leukemia line.                                         

EXAMPLE 3

Inhibition of Tumor Cell Growth In Vitro by hCG

In this Example, we demonstrate that hCG inhibits growth of Kaposi'ssarcoma cells in vitro. We tested the ability of the Kaposi's sarcomacell line KS Y-1 to form colonies in methylcellulose in the presence ofhCG. Four different forms of hCG were tested: intact native hCG(includes both α- and β-subunits), native β-hCG, β-hCG.sub.(109-145) (afragment consisting of amino acids 109-145 of β-hCG), and α-hCG.

We plated KS Y-1 or control cells (5×10⁵ cells/ml) in methylcellulose,using the protocol of Lunardi-lskandar et al. (1985) Clin. Exp. Immunol.60: 285-293. Cells were incubated at 37° for 48 hr, after which colonieswere counted. Results are shown in Table 3, expressed as number ofcolonies per 5×10⁴ cells plated. Concentrations of hCG are in μg/ml.

Native hCG, the β-subunit of hCG alone, and βhCG.sub.(109-145) allinhibited growth of KS Y-1 cells in a dose-dependent manner. βhCG andβhCG.sub.(109-145) completely inhibited colony formation at 10 μg/ml.αhCG caused, at most, a slight decrease in number of colonies formed.None of the hCG preparations inhibited colony formation by the controladult T-cell leukemia cell line (ATL 1) or peripheral blood lymphocytes(PBL).

                  TABLE 3                                                         ______________________________________                                        Both hCG or βhCG block KS Y-1 cell growth in a methylcellulose           clonogenic assay.                                                             Treatment (μg/ml hCG)                                                                   βhCG                                                                hCG  native     βhCG.sub.109-145                                                                   αhCG                                  Cells None    12.5   1    10    1    10   1     10                            ______________________________________                                        KS Y-1                                                                              234      10     24   0     34   0   224   179                           ATL 1 310     267    289  267   299  278  298   285                           PBL   320     319    287  290   301  315  818   330                           ______________________________________                                         KS Y1 cells were seeded in methylcellulose in the presence of hCG, native     βhCG, βhCG synthetic peptide.sub.109-145 or αhCG. The hCG     preparation contains both α- and β-hCG. ATL1 is a human adult      Tcell leukemia line. Numbers indicate number of colonies per 50,000 cells     plated.                                                                  

hCG also inhibits the uptake of ³ H-thymidine into Kaposi's sarcomacells. This provides further demonstration of the inhibitory effect ofhCG on KS cell proliferation. The cells were cultured in flat bottomed96-well tissue culture plates (3072, Falcon Labware) at various celldensities in RPMI1640+FCS medium in the presence or absence of hCG.Proliferation was evaluated by a 4 hr pulse of ³ H-thymidine (Amersham,Arlington Heights Ill.) added at 48 hr after culture initiation.Thymidine incorporation was reduced by about 80% in cells grown in thepresence of 10 and 100 USP units hCG per ml.

EXAMPLE 4

hCG Inhibits Tumorigenesis and Metastasis in vivo

To demonstrate that hCG inhibits tumorigenesis and metastasis in vivo,we injected KS Y-1 cells that had been treated with KS Y-1 intoimmunodeficient mice. We grew KS Y-1 cells in RPMI 1640 mediumcontaining 10% fetal calf serum, 1% penicillin/streptomycin, and 1%glutamine for 48 hr in the presence of 10 USP units hCG per ml. Thetreated cells (5×10⁵) were then injected into Bg-Nude mice.

For two months post-injection, mice were analyzed for the presence oftumors. Results are presented in Table 4. Each of the control mice(injected with cells that had not been pretreated with hCG) had tumors,while no tumors were observed in the mice that received hCG-treated KSY-1 cells. We observed metastasis in the control mice, as tumors aroseat sites other than the site of injection.

                  TABLE 4                                                         ______________________________________                                        In vitro pretreatment of KS Y-1 cells With hCG inhibits angiogenesis          and metastasis in Bg-nude mice.                                               Cell treatment                                                                          Tumor size (mm)                                                                            Angiogenesis                                                                              Metastasis                                 ______________________________________                                        No treatment                                                                            17 × 22 to 27 × 30                                                             +           +                                          (n = 7)                                                                       hCG source A                                                                            0*           -           -                                          (n = 6)                                                                       hCG source B                                                                            0*           -           -                                          (n = 6)                                                                       ______________________________________                                         5 × 10.sup.6 KS Y1 cells were treated for 48 h with control (PBS) o     10 USP units/ml hCG from source A (Sigma: CG10) or source B (Ayerst Lab:      APL ™) before subcutaneous injection into Bgnude mice. Animals were        sacrificed at 6 weeks post inoculation.                                       n = number of animals in test group.                                          *No tumors were observed by macroscopic or light microscopic evaluation o     the injection site within 6 weeks postinjection.                         

EXAMPLE 5

hCG Pretreatment of Mice Inhibits Tumorigenesis

To further demonstrate that hCG inhibits tumorigenesis in vivo, weinjected mice with hCG prior to challenge with Kaposi's sarcoma cells.Mice were injected with hCG (100 USP units) daily for seven days. Afterthis pretreatment, 10⁵ KS Y-1 cells in 0.1 ml were injectedsubcutaneously or intraperitoneally into each mouse. The mice wereexamined for presence of tumors, angiogenesis and metastasis at 3-4weeks post inoculation.

The results of these experiments are shown in Table 5. Half of the micethat had been pretreated with hCG did not develop tumors, while thetumors that arose in the remaining pretreated mice were much smallerthan the tumors found in mice that had not been pretreated.

                  TABLE 5                                                         ______________________________________                                        In vivo pretreatment of immunodeficient mice (Balb/c nude) with hCG           inhibits tumorigenesis induced by KS Y-1 cells.                               Treatment, Tumor size                                                         administration                                                                           (mm)        Angiogenesis                                                                             Metastasis                                  ______________________________________                                        Human hCG  0 × 0                                                        source A,  0 × 0                                                        subcutaneous                                                                             0 × 0                                                        (n = 6)    2 × 3 +          -                                                      1 × 2 +          -                                                      2 × 2 +          -                                           Human hCG  0 × 0                                                        source B,  0 × 0                                                        intraperitoneal                                                                          0 × 0                                                        (n = 6)    1 × 1 +          -                                                      1 × 1 +          -                                                      2 × 1 +          -                                           No treatment,                                                                            45 × 35                                                                             +          +                                           subcutaneous                                                                             41 × 19                                                                             +          +                                           (n = 4)    42 × 39                                                                             +          +                                                      39 × 37                                                                             +          +                                           ______________________________________                                         Animals were pretreated with hCG 100 USP units (IP or SC) daily for 1 wee     and then injected with KS Y1, 10.sup.5 cells in 0.1 ml. Source A (Sigma:      CG10), source B (Ayerst Lab: APL ™). The "No treatment" controls           consisted of injections of PBS.                                          

EXAMPLE 6

hCG Induces Tumor Regression in vivo

In this Example, we demonstrate that hCG treatment causes regression ofestablished tumors. We intralesionaly or intraperitoneally injected 100USP units hCG daily for 7 days into mice that had established,metastatic tumors. Tumors had been induced by inoculatingimmunodeficient mice with KS Y-1 Kaposi's sarcoma cells. Within twoweeks after the one week treatment regime ended, the tumors had shrunkensignificantly, and appeared necrotic. Untreated mice had large,metastatic tumors, and we observed no necrosis of the tumor cells.

To determine the mechanism by which hCG inhibits tumor growth, wecompared tumor tissues from mice that had been treated with hCG or β-hCGto mice that had not been treated. Tumor tissues were collected at leastone month after inoculation with the Kaposi's sarcoma cell line.Microscopic examination of βhCG-treated tumors showed focal areascontaining few cells, the absence of edema, and necrosis. Theseobservations suggested that the tumor cells had been eliminated by anapoptotic process.

To confirm that tumor regression occurred by an apoptotic mechanism, westained slides of tissues from hCG-treated tumors to detect the presenceof DNA fragmentation (DNA laddering) that is characteristic of apoptosisCohen (1991) Adv. Immunol. 50: 55-85 (1991); Clarke et al. (1990) Anat.Embryol. 181: 195-213)!. For this analysis, we employed the ApopTag™(Oncor, Inc., Gaithersburg, Pa.) in situ apoptosis detection kitaccording to the instructions supplied by the manufacturer (which areincorporated herein by reference). More than 90% of the cells in regionsof the tumors that were regressing were positive for DNA laddering (FIG.1).

Ninety percent of the tumors from animals treated with hCG expressedgenes that are indicative of apoptosis c-myc, Cohen (1991) Adv. Immunol.50: 55-85; c-rel, Abbabie et al. (1993) Cell 75: 899-912!. We alsodetected the polypeptide gene product of this gene. Tumor cells fromanimals that had not been treated with hCG did not express thisapoptosis gene. Therefore, hCG appears to induce apoptosis in tumorcells.

EXAMPLE 7

hCG Receptor Present Kaposi's Sarcoma Cells

We utilized radiolabelled hCG in chemical crosslinking experiments todetect the presence of the hCG receptor in various cell types. We testedcell lysates of primary cells from the pleural effusion of five Kaposi'ssarcoma patients (Patients #1-4), a colon carcinoma patient (#1539),cell line KS Y1 p 35 and strain KS 4 p9 (See FIG. 2). As a positivecontrol we used colon carcinoma hepatoma cell lines and as a negativecontrol we used peripheral blood lymphocytes from normal adults.

The binding of ¹²⁵ I-hCG to the hCG receptor was performed as describedby Sahraoui et al. (1992) Cellular Immunol. 139: 318-332. The hCG andreceptor were cross-linked by incubating a pellet of 5×10⁶ cells in 50μl of dimethylsulfoxide in which we had diluted 1 μl of 1 mg/mldisuccinimidyl suberate solution (DSS) in dimethyl sulfoxide. Afterincubating for 10 min. at 4° C., the reaction is stopped by quenchingthe unreacted DSS with 10 μl of a 1 mM ammonium acetate solution for oneminute. The cells are then washed twice at 4° C. in 10 mM Tris-HCl, pH7.4, 0.15M NaCl containing 1 μM EDTA. The crosslinked ¹²⁵ I-hCG-receptorwas released from the cells by extracting for 30 min at 4° C. in 0.5%NP40 in 0.05M Tris-HCl, pH 7.4, 0.15M NaCl containing 50 μg/mlaprotinin, 50 μg/ml leupeptin, 0.1% sodium azide, and 10 mM sodiumpyrophosphate. The lysates were clarified by centrifugation (15 kRPM for15 min. at 4° C.) and size-fractionated on a 6% SDS-polyacrylamide gel.

We detected a 70 kD band that coincides with the M, of β-hCG coupled toits receptor Hipkin et al. (1993) Mol. Endocrinol. 29: 121-125! (FIG.2). We also tested KS lesions from eight AIDS patients byimmunostaining, and detected the presence of the hCG receptor in alleight lesions. These results strongly suggest that the antitumor effectof β-hCG may be a direct consequence of hCG interaction with itsreceptor, leading to apoptotic cell death.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications and patent documentsreferenced in this application are incorporated herein by reference.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

What is claimed is:
 1. An article of manufacture comprising packagingmaterial and a pharmaceutical agent contained within the packagingmaterial, wherein the pharmaceutical agent is therapeutically effectivefor treating Kaposi's sarcoma and is selected from the group consistingofi. human chorionic gonadotropin (hCG); ii. a β-subunit of hCG and iii.a biologically active fragment of hCG or a β-subunit of hCG; and whereinthe packaging material comprises a label which indicates that about 3000to about 5000 United States Pharmacopeia ("USP") units per 70 kg bodyweight per day of the pharmaceutical agent is useful for treatingKaposi's sarcoma.
 2. A method for treating Kaposi's sarcoma in apatient, the method comprising administering to the patient about 3000to about 5000 United States Pharmacopeia ("USP") units per 70 kg bodyweight per day of a pharmaceutical agent selected from the groupconsisting ofi. human chorionic gonadotropin (hCG); ii. a β subunit ofhCG; and iii. a biologically active fragment of hCG or a β-subunit ofhCG.
 3. An article of manufacture as in claim 1 wherein the label statesthat the agent should be administered to a patient to maintain its invivo concentration at between 100 and 200 United States Pharmacopeia("USP") units per ml of plasma.
 4. An article of manufacture as in claim3 wherein the label states that the plasma lever of the agent should beadministered to a patient to maintain its in vivo concentration at about160 USP units per ml.
 5. A method of claim 2 wherein the agent isadministered in an amount sufficient to maintain the concentration ofthe agent in the patient's plasma between 100 and 200 United StatesPharmacopeia ("USP") units per ml of plasma.
 6. A method of claim 2wherein the agent is β-hCG(109-145).
 7. A method of claim 2 wherein theagent is β-hCG(109-119).